苦参碱介导上皮间质转化逆转肺癌A549顺铂耐药株活性研究

Reversal of Cisplatin Resistance in Lung Cancer A549 Cells by Matrine-mediated Cell Epithelial-mesenchymal Transition

目的探讨苦参碱(MT)逆转肺癌A549顺铂耐药株活性中的作用及分子机制。方法对数生长期人肺腺癌耐顺铂细胞株A549/DDP细胞,用不同浓度MT(1,4,16,32μmol·L^(-1))处理,采用噻唑蓝(MTT)法在24 h检测细胞增殖抑制率。将细胞分为空白对照组及MT组(16μmol·L^(-1)),光学显微镜、Transwell及免疫荧光检测两组细胞的细胞形态、侵袭能力、E-钙粘蛋白(E-cadherin)及波形蛋白(vimentin)细胞免疫荧光变化。另将细胞分为空白对照组、MT组(16μmol·L^(-1)MT)、顺铂(DDP)组(3×10^(-3)μmol·L^(-1)DDP)和MT+DDP组(16μmol·L^(-1)MT+3×10^(-3)μmol·L^(-1)DDP),Western blotting法检测上皮间充质转化(EMT)表型相关标志物E-caherin、vimentin、slug、p-p65等蛋白表达,流式细胞仪检测细胞凋亡率。结果MT能明显抑制A549/DDP细胞的增殖及侵袭能力,MT处理后细胞呈现上皮细胞样形态及上皮表型。此外,与对照组及DDP组比较,MT+DDP组细胞凋亡率明显升高(P<0.05),E-cadherin蛋白表达水平明显升高(P<0.05),vimentin、slug及p-p65蛋白表达水平明显降低(P<0.05)。结论苦参碱具有逆转肺癌顺铂耐药的作用,其机制与抑制肺癌细胞上皮间质转化及抑制核因子NF-κB信号通路有关。

Objective To investigate the role and molecular mechanism of matrine(MT)in reversing cisplatin(DDP)resistant lung cancer cell A549 in vitro.Methods Cisplatin resistant cell line A549/DDP cells in the logarithmic growth phase were treated with different concentrations of MT(1,4,16,and 32μmol·L^(-1)).Subsequently,MTT method was applied to detect cell proliferation inhibition rate at 24 h.The cells were divided into blank control group and MT group(16μmol·L^(-1)),and fluorescence microscopy,and Western blotting analyses were used to detect the changes of cell morphology,invasion ability,E-cadherin and vimentin cell immunofluorescence.Changes in EMT markers after drug treatments were assayed in four groups of cell settings,blank control control(A549/DDP cell),MT group(A549/DDP+16μmol·L^(-1)matrine),DDP group(A549/DDP+3×10^(-3)μmol·L^(-1)DDP),and MT+DDP group(A549/DDP+16μmol·L^(-1)matrine+3×10^(-3)μmol·L^(-1)DDP).The lung cancer cisplatin-resistant subline A549/DDP were treated with matrine,cisplatin and their combination for cell apoptosis and Western blotting analyses.Results MT could significantly inhibit the proliferation and invasion of A549/DDP cells.After MT treatment,the cells showed epithelial cell like morphology and epithelial phenotype.In addition,compared with blank control group and DDP group,the apoptosis rate of MT+DDP group was significantly higher(P<0.05),and the expression level of E-cadherin protein was significantly higher(P<0.05),and the expression levels of vimentin,slug and p-p65 protein were significantly lower(P<0.05).Conclusion Matrine treatment was able to reverse cisplatin resistance in lung cancer,and its mechanism was related to the inhibition of epithelial mesenchymal transformation and inactivation of the NF-κB signaling.